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Mode of Action (MoA)
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binds to Cas9, blocking DNA binding.
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Evidence
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AcrIIC4Hpa was identified as a Cas9 inhibitor through in vitro cleavage assays using recombinant NmeCas9 and sgRNA-loaded complexes. Addition of AcrIIC4Hpa blocked target DNA cleavage in a concentration-dependent manner, with full inhibition observed at approximately 20-fold molar excess. In E. coli phage Mu immunity assays, AcrIIC4Hpa expression rescued phage infectivity in strains expressing HpaCas9 (its cognate Cas9) and significantly reduced NmeCas9-mediated phage targeting, confirming in vivo inhibition. Co-purification assays demonstrated direct binding between AcrIIC4Hpa and both HpaCas9 and NmeCas9. In HEK293T cells, transient coexpression of AcrIIC4Hpa abolished NmeCas9 genome editing activity without affecting SpyCas9, showing subtype specificity. Electrophoretic mobility shift assays (ECO_0000096) revealed that AcrIIC4Hpa does not interfere with sgRNA loading but blocks target DNA binding, consistent with a mechanism that prevents stable Cas9-DNA association.
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MoA Category
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binds and inhibits host defence system
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Subtype(s) of the defence system(s) inhibited by the protein
Defence Subtype
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Neisseria meningitidis type II-C CRISPR-Cas
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Relevant publication(s)
DOI
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10.1128/mBio.02321-18
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Other components of the anti-defence system
Multicomponent System
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-
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Known structure in PDB
PDB ID
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7F7P_A
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Genome(s) encoding the protein
Protein Source
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MGE in Haemophilus parainfluenza
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Defence system(s) inhibited by the protein
Defences
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CRISPR-Cas
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